4123
J. Org. Chem. 1984, 49, 4123-4127
Designing Photoaffinity Labeling Reagents for Chromatin Studies Ole Buchardt,*pt Ulrich Ehrbar,t Charles Larsen,t Jargen Maller,t Peter E. Nielsen,! Tove Thomsen,+Frank Watjen,+and John Bondo Hansent Chemical Laboratory ZZ, The H.C. 0rsted Institute, University of Copenhagen, Universitetsparken 5, DK-2200 Copenhagen, Denmark, Department of Chemistry, University of Odense, Campusvej 55, DK-5230 Odense M, Denmark, and Biochemical Institute B, The Panum Institute, Blegdamsvej 3, DK-2200 Copenhagen N , Denmark Received December 28, 1983
The synthesis of a series of reagents for photoaffinity labeling of the proteins in chromatin is described and discussed. The reagents contain one or two DNA-intercalating groups, a photoprobe, and a fluorometrically detectable group. The syntheses were aimed at supplying the reagents with basic groups. Furthermore, eventual radiolabeling of reagents should be possible. The two intercalating groups are connected by a linker, and the synthetic strategy allows this linker, a~ well as the linker to the photoprobe, to be variable in length and molecular design. 9-Aminoacridine moieties serve as intercalaton, and aliphatic amino or guanidino groups serve as assembly points in the synthesis, as well as the warranted basic groups.
Chromatin, which contains the genetic material of all eucariotic organisms, consists of DNA, five characteristic basic proteins, i.e., the histones, and a large number of little understood, and poorly characterized, so-called nonhistone chromosomal pr0teins.l The structure and function of these, and their interaction with DNA, are currently the subject of intense study by many groups,lJ and photoaffinity labeling is one among several methods which can be employed to obtain new information about the interaction between DNA and protein in chromatin. We are currently employing the technique"* and report here the synthetic approach toward a series of new reagents for this purpose. The synthetic strategy was thus directed toward compounds which should (1) have strong and specific reversible binding to double-helixed DNA, (2) contain a photoprobe, i.e., a molecular moiety which is unreactive until irradiated (A > 300 nm), whereupon it can be irreversibly bound, preferentially to protein, in situ, (3) contain a label which ensures detectability upon very large dilution, e.g., fluorescence or radioactivity. Furthermore, the synthetic strategy should ensure that the intramolecular distances between the groups necessary to obtain the above qualities should be variable (variable linkers), and these groups should themselves be variable. Finally, a number of secondary qualities of the reagents would be needed, e.g., solubility in aqueous solvents, sufficient thermal stability, etc. We decided to employ the following molecular design: (1)Strong and reversible binding to DNA should be ensured by using intercalators, i.e., polycyclic aromatic groups such as 9-amin~acridines~ or ethidium: and eventually by including basic groups in the reagents, which under physiological conditions would be positively charged. This should result in further binding to the negatively charged phosphate backbone of the DNA. By employing two intercalating groups, superior DNA binding could be obtained, and by varying the intercalators, some degree of site specificity could be obtained. (2) A variety of photoprobes' should be employable, and aryl azides were chosen for the present work. (3) If 9-aminoacridines or ethidium groups were employed, fluorescence detection would be pwqible, or the basic groups could be rendered radioactive upon methylation with tritiated methyl iodide, etc. Furthermore, the potential use of a radioactive linker was included in the synthetic scheme. + T h e H. C. 0rsted Institute. *University of Odense. $ T h e Panum Institute.
Scheme I
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Scheme I1
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